The relationship between regional adipose tissue (AT) distribution and metabolic diseases has led to an interest in quantifying regional Glycerol (GLY) kinetics. We studied regional GLY kinetics in 6 male subjects (age 36+/-3 yr). An abdominal vein draining subcutaneous (SQ) adipose tissue (AT) and a radial artery were cannulated. Four microdialysis probes were inserted into interstitial SQ AT. A primed-constant infusion of [2H5]glycerol was infused for 120 min and abdominal AT blood flow was determined by 133xenon washout. GLY tracer:tracee ration in the artery was greater thatn in the vein which was greater than in interstitial fluid. Plasma GLY concentrations in abdominal vein was higher than in the artery. A plateau in GLY isotope enrichment was achieved by 45 minutes of infusion in both artery and abdominal vein samples. Tracer balance across adipose tissue was not significantly different from zero. Values for regional GLY production calculated by tracer methodology and be arteriovenous balance techniques were similar with good correlation between the two methods. We conclude the (1) a primed constant infusion of GLY tracer reaches isotopic steady state by 45 min, (2) no net extraction of GLY tracer occurs across AT, (3) GLY in AT interstitial fluid should be considered a distinct pool, and (4) regional GLY kinetics in AT can be described by at least a 3-compartment model consisting of arterial, venous, and interstitial pools.